Citrus peel extract as 3-hydroxy-3-methylglutaryl CoA(HMG-CoA) reductase inhibitor

ABSTRACT

A method for inhibiting the activity of 3-hydroxy-3-methylglutaryl CoA(HMG-CoA) reductase in mammals comprises administering citrus peel extract thereto.

FIELD OF THE INVENTION

The present invention relates to a method for inhibiting the activity of3-hydroxy-3-methylglutaryl CoA(HMG-CoA) reductase in a mammal whichcomprises administering citrus peel extract thereto.

BACKGROUND OF THE INVENTION

In recent years, coronary cardio-circulary diseases, e.g.,atherosclerosis and hypercholesterolemia, have increasingly become amajor cause of deaths. It has been reported that an elevated plasmacholesterol level causes the deposition of fat, macrophages and foamcells on the wall of blood vessels, such deposit leading to plaqueformation and then to atherosclerosis(Ross, R., Nature, 362,801-809(1993)). One of the methods for decreasing the plasma cholesterollevel is alimentotherapy to reduce the ingestion of cholesterol andlipids. Another method is to lower the rate of cholesterol biosynthesiswhich takes place in the liver. It has been reported thathypercholesterolemia can be treated effectively by reducing the rate ofcholesterol biosynthesis through the inhibition of HMG-CoA reductasewhich mediates the synthesis of mevalonic acid, an intermediate in thebiosynthesis of sterols or isoprenoids (Cardiovascular Pharmacology,William W. Parmley and Kanu Chatterjee Ed., Wolfe Publishing, pages8.6-8.7, 1994).

Therefore, numerous efforts have been made to develop medicines toinhibit HMG-CoA reductase; and, as a result, several compounds derivedfrom Penicillium sp. and Aspergillus sp. have been commercialized.Specifically, Lovastatin® and Simvastatin® developed by Merck Co.,U.S.A., and Pravastatin® developed by Sankyo Co., Japan, have beencommercialized(C. D. R. Dunn, Stroke: Trends, Treatment and Markets,SCRIPT Report, PJB Publications Ltd., 1995). However, these medicinesare very expensive and a long-term administration thereof is known toinduce an adverse side effect of increasing creatine kinase in theliver. Accordingly, there has continued to exist a need to develop aninexpensive and non-toxic inhibitor of HMG-COA reductase.

Citrus peel has been used as digestion improving agent. However, therehas been no report on the HMG-COA reductase inhibitory activity of thecitrus peel extract.

SUMMARY OF THE INVENTION

Accordingly, it is a primary object of the present invention to providea method for inhibiting the HMG-COA reductase activity in mammals.

In accordance with one aspect of the present invention, there isprovided a method for inhibiting the HMG-CoA reductase activity inmammals which comprises administering citrus peel extract thereto.

DETAILED DESCRIPTION OF THE INVENTION

The method of the present invention for inhibiting the activity ofHMG-CoA in mammal comprises administering citrus peel extract thereto.

The citrus may be tangerines, oranges, lemons, grapefruits and Poncirustrifoliata.

The citrus peel extract may be prepared by any of the conventionalmethods using alcohols or water. For instance, 5 to 100 l alcohol orwater is added to 1 kg of dried citrus peel and the mixture is allowedto stand at a temperature ranging from 5° to 80° C. for alcohol or from20° to 140° C. for water for a period ranging from 1 to 48 hours. Thisextraction process may be repeated 1 to 3 times. The resulting extractis concentrated, e.g., by vacuum, to obtain a concentrated peel extract.Further, it can be prepared by treating citrus peels with calciumhydroxide solution, adding hydrochloric acid thereto to adjust solutionto a pH ranging from 4.0 to 4.5, and then centrifuging the resultingsolution to obtain the precipitates, flavonoides, as citrus peel extract(FDA Report No. FDA-BF-82/62, "Evaluation of health aspects ofhesperidin, naringin and citrus bioflavonoide extracts as foodingredient", Natl. Technical Information Service PB 82-192931(1982)).

The citrus peel extract exerts an inhibitory effect on the HMG-COAreductase at a dose of 10 mg/kg/day or more, the inhibitory effectincreasing with the dose thereof.

Moreover, in spite of their potent efficacies, the citrus peel extractshows little toxicity or mitogenicity in tests using mice. Morespecifically, the citrus peel extract exhibits no toxicity when it isorally administered to a mouse at a dosage of 1,000 mg/kg, whichcorresponds to oral administration dose of 50 to 100 g/kg body weight ofcitrus peel extract for a person weighing 50 kg. Further, the citruspeel extract exerts no adverse effects on the liver function.

The present invention also provides a pharmaceutical composition forinhibiting the HMG-COA reductase activity, which comprises citrus peelextract as an active ingredient, in combination with pharmaceuticallyacceptable excipients, carriers or diluents.

A pharmaceutical formulation may be prepared by using the composition inaccordance with any of the conventional procedures. In preparing theformulation, the active ingredient is preferably admixed or diluted witha carrier, or enclosed within a carrier which may be in the form of acapsule, sachet or other container. When the carrier serves as adiluent, it may be a solid, semi-solid or liquid material acting as avehicle, excipient or medium for the active ingredient. Thus, theformulations may be in the form of a tablet, pill, powder, sachet,elixir, suspension, emulsion, solution, syrup, aerosol, soft and hardgelatin capsule, sterile injectable solution, sterile packaged powderand the like.

Examples of suitable carriers, excipients, and diluents are lactose,dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, alginates,gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water,methylhydroxybenzoates, propylhydroxybenzoates, talc, magnesium stearateand mineral oil. The formulations may additionally include fillers,anti-agglutinating agents, lubricating agents, wetting agents, flavoringagents, emulsifiers, preservatives and the like. The compositions of theinvention may be formulated so as to provide quick, sustained or delayedrelease of the active ingredient after their administration to a mammalby employing any of the procedures well known in the art.

The pharmaceutical formulation of the present invention can beadministered via various routes including oral, transdermal,subcutaneous, intravenous and intramuscular introduction. In case ofhuman, a typical daily dose of citrus peel extract may range from about10 to 500 mg/kg body weight, preferably 20 to 100 mg/kg body weight, andcan be administered in a single dose or in divided doses. However, itshould be understood that the amount of the active ingredient actuallyadministered ought to be determined in light of various relevant factorsincluding the condition to be treated, the chosen route ofadministration, the age, sex and body weight of the individual patient,and the severity of the patient's symptom; and, therefore, the abovedose should not be intended to limit the scope of the invention in anyway.

Moreover, the citrus peel extract can be incorporated in foods orbeverages for the purpose of inhibiting the HMG-CoA reductase activity.

As described above, the citrus peel extract can be used as an effective,non-toxic pharmaceutical agent for inhibiting HMG-CoA reductaseactivity. In this case, the content of the citrus peel extract in a foodor beverage may range from 0.1 to 50%.

The following Examples are intended to further illustrate the presentinvention without limiting its scope.

Further, percentages given below for solid in solid mixture, liquid inliquid, and solid in liquid are on a wt/wt, vol/vol and wt/vol basis,respectively, and all the reactions were carried out at roomtemperature, unless specifically indicated otherwise.

EXAMPLE 1 Preparation and Analysis of Citrus Peel Extract

Citrus peels were dried at room temperature and to 6.7 kg of the driedpeels was added 80 l of 95% ethanol. The mixture was allowed to stand atroom temperature for 24 hours and filtered to obtain an extract and asolid residue. The solid residue was extracted one more time by the sameprocedure. The extract thus obtained were combined, concentrated under areduced pressure using a large capacity evaporator (EYELA Rotary vacuumevaporator N-11) to obtain 1.7 kg of concentrated extract (d=1.3 g/ml).

The concentrated extract was diluted in DMSO/methanol(1:1) mixture at aconcentration of 10 mg/ml. 100 μof the resulting solution was subjectedto high performance liquid chromatography (HPLC) using PhenomenexProdigy column (5μ ODS(3) 100Å, 4.6×250 mm) which was pre-equilibratedwith 0.01M phosphoric acid/methanol (70:30) mixture. The sample waseluted with 0.01M phosphoric acid/methanol(70:30) mixture for 55 min.with increasing gradually the concentration of methanol to 45% at a flowrate of 0.6 ml/min. 100 μl (1 mg/ml) of hesperidin and naringin(SigmaChemical Co., USA) were used as standard materials. The eluates weredetected at 280 nm and it was discovered that lkg of the citrus peelextract contains 5,950 mg of hesperidin and 280 mg of naringin.

Further, ingredients of the citrus peel extract were identified inaccordance with a conventional method. For instance, moisture contentwas determined by dry method at 105° C.; crude protein, by Kjeldahlmethod; crude lipid, by Soxhlet method; free saccharide, by Bertrandmethod; crude ash, by ashing at 550°-600° C.; and hesperidin andnaringin, by HPLC method. The result is shown in Table I.

                  TABLE I                                                         ______________________________________                                        Ingredient       Content (%)                                                  ______________________________________                                        Moisture         39.1                                                         Crude protein    2.7                                                          Crude lipid      1.8                                                          Free        Fructose 20.0                                                     saccharide  Glucose  16.5                                                                 Sucrose  8.6                                                      Crude ash        1.0                                                          Hesperidin       0.6                                                          Naringin         0.03                                                         Other saccharides                                                                              9.67                                                         ______________________________________                                    

EXAMPLE 2 Administration of Citrus Peel Extract to an Animal

20 four-week-old Sprague-Dawley rats (Taihan laboratory animal center,Korea) each weighing about 90 to 110 g were evenly divided into twodietary groups by a randomized block design. The rats of the two groupswere fed with two different high-cholesterol diets, i.e., AIN-76laboratory animal diet (ICN Biochemicals, Cleveland, Ohio U.S.A.)containing 1% cholesterol(Control group), and 1% cholesterol plus 16.7%citrus peel extract, respectively. The compositions of diets fed to thetwo groups are shown in Table II.

                  TABLE II                                                        ______________________________________                                                        Dietary group                                                                           Citrus peel                                                           Control extract*.sup.2                                      Component         group   group                                               ______________________________________                                        Casein            20      20                                                  D,L-methionine    0.3     0.3                                                 Corn starch       15      15                                                  Sucrose           49      32.3                                                Cellulose powder*.sup.1                                                                         5       5                                                   Mineral mixture*.sup.1                                                                          3.5     3.5                                                 Vitamin mixture*.sup.1                                                                          1       1                                                   Choline citrate   0.2     0.2                                                 Corn oil          5       5                                                   Cholesterol       1       1                                                   Citrus peel extract       16.7                                                Total             100     100                                                 ______________________________________                                         *.sup.1 Purchased from TEKLAD premier Co. (Madison, WI, U.S.A.)               *.sup.2 0.1% hesperidin equivalent                                       

The rats were allowed to feed freely on the specified diet together withwater for six weeks, the ingestion amount was recorded daily and therats were weighed every 7 days, and then the record was analyzed. Allrats showed a normal growth rate and there was observed no significantdifference among the two groups in terms of the feed ingestion amountand the weight gain.

EXAMPLE 3 Determination of Total Cholesterol, HDL- cholesterol andNeutral Lipid Content in plasma

The effect of administering citrus peel extract to rats on the plasmacholesterol and neutral lipid content was determined as follows.

Blood samples were taken from the rats of the two dietary groups andplasma HDL fractions were separated therefrom by using HDL-cholesterolreagent (Sigma Chemical Co., Cat. No. 352-3) containing dextran-sulfate.Total cholesterol and HDL-cholesterol levels were determined by usingSigma Diagnostic Kit Cat. No. 352-100(Sigma Chemical Co., U.S.A.)(Allain et al., Clin. Chem. , 20, 470-475(1974)). Neutral lipids levelwas determined by using Sigma Diagnostic Kit Cat. No. 339-50(Bucolo, G.and David, H., Clin. Chem., 19, 476-482(1973)). The result is shown inTable III, wherein the total plasma cholesterol level decreased by 35%in the citrus peel extract-fed rat group, as compared with that of thecontrol group.

                  TABLE III                                                       ______________________________________                                                     Group                                                                                     Citrus peel                                                         Control   extract                                              Lipids Conc.   group     group                                                ______________________________________                                        Total-C (mg/dl)                                                                              147.8 ± 34.8                                                                         94.2 ± 23                                         HDL-C (mg/dl)  22.2      23.5                                                  ##STR1##      15.7 ± 5.3                                                                           26.2 ± 7.5                                        TG (mg/dl)      99.2 ± 18.9                                                                         108.5 ± 15.9                                      Atherosclerosis                                                                               6.3 ± 3.4                                                                            3.1 ± 1.2                                        index                                                                         ______________________________________                                         *Total-FC: Totalcholesterol                                                   *HDLC: HDLcholesterol                                                         *TG: Triglyceride                                                        

EXAMPLE 4 Activity of Citrus Peel Extract in HMG- COA ReductaseInhibition

(Step 1) Preparation of microsomes

To determine the effect of citrus peel extract feeding to rats on theactivity of HMG-COA reductase, a regulatory enzyme of the cholesterolsynthesis in the liver, microsomes were separated from the liver tissueto be used as an enzyme source.

First, the rats of the two groups were sacrificed by decapitation andthe livers were excised and immediately placed in an ice-coldhomogenization medium(50 mM KH₂ PO₄ (pH 7.0), 0.2M sucrose, 2 mMdithiothreitol (DTT)). The livers were homogenized in the homogenizationmedium (2 ml medium/g of the liver) with a Waring blendor for 15sec.(three strokes with a motor-driven Teflon pestle in aPotter-Elvehjem type glass homogenizer). The homogenate was centrifugedat 15,000×g for 10 min. and the supernatant thus obtained wascentrifuged at 100,000×g for 75 min. to obtain microsomal pellets, whichwere then resuspended in the homogenization medium containing 50 mM EDTAand centrifuged at 100,000×g for 60 min. The supernatant containing themicrosome was used as an enzyme source.

(Step 2) HMG-CoA reductase assay

The activity of HMG-CoA reductase was determined by employing ¹⁴C!HMG-CoA, in accordance with the method of Shapiro et al.(Biochemica etBiophysica Acta, 370, 369-377(1974)) as follows.

The enzyme in the supernatant containing the microsome obtained in(Step 1) was activated at 37° C. for 30 min. Added to a reaction tubewere 20 μl of HMG-COA reductase assay buffer (0.25M KH₂ PO₄ (pH 7.0),8.75 mM EDTA, 25 mM DTT, 0.45M KCl and 0.25 mg/ml BSA), 5 μl of 50 mMNADPH, 5 μl of ¹⁴ C!HMG-CoA (0.05 μCi/tube, final conc. 120 μM), and 10μl of activated microsomal enzyme (0.03-0.04 mg), and the mixture wasincubated at 37° C. for 30 min. The reaction was terminated by adding 10μl of 6M HCl to the mixture, and the mixture was incubated at 37° C. for15 min. to allow complete lactonization of the product (mevalonate). Theprecipitate was removed by centrifugation at 10,000×g for 1 min. and thesupernatant was applied to a Silica gel 60 G TLC plate (Altech, Inc.,Newark, U.S.A.) and then developed with benzene:acetone(1:1, v/v). Aregion having a Rf value ranging from 0.65 to 0.75 was removed byscraping with a disposable cover slips and assayed for radioactivitywith 1450 Microbeta liquid scintillation counter (Wallacoy, Finland).Enzyme activities were calculated as picomoles mevalonic acidsynthesized per min. per mg protein (pmoles/min/mg protein). The resultis shown in Table IV.

                  TABLE IV                                                        ______________________________________                                                       Group                                                                                 Citrus peel                                                           Control extract                                                               group   group                                                  ______________________________________                                        HMG-CoA reductase                                                                              147 ± 12.5                                                                           112.1 ± 12.8                                    activity                                                                      (pmole/min/mg protein)                                                        ______________________________________                                    

As can be seen from Table IV, the control group rats showed a relativelyhigh HMG-CoA reductase activity, while the HMG-CoA activity observedwith the citrus peel extract- fed rat group is lower than that of thecontrol group by 34%.

EXAMPLE 5 Toxicity of Orally Administered Citrus Peel Extract

7 to 8 week-old, specific pathogen-free ICR female mice (8 heads) eachweighing about 25 to 29 g and male mice (8 heads) each weighing about 34to 38 g were bred under a condition of temperature 22°±1° C., moisture55±5% and photoperiod 12L/12D. Fodder (Cheiljedang Co., mouse and ratfodder) and water were sterilized and fed to the mice.

The citrus peel extract was dissolved in 0.5% Tween 80 to aconcentration of 100 mg/ml, and the solution was orally administered tothe mice in an amount of 0.2 ml per 20 g of mouse body weight. Thesolution was administered once and the mice were observed for 10 daysfor signs of adverse effects or death according to the followingschedule: 1, 4, 8, and 12 hours after the administration and, every 12hours thereafter. The weight changes of the mice were recorded every dayto examine the effect of citrus peel extract. Further, on the 10th day,the mice were sacrificed and the internal organs were visually examined.

All the mice were alive at day 10 and citrus peel extract showed notoxicity at a dose of 1,000 mg/kg. The autopsy revealed that the micedid not develop any pathological abnormality, and no weight loss wasobserved during the 10 day test period. Accordingly, it was concludedthat the citrus peel extract is not toxic when orally administered to ananimal.

The following Formulation Example is for illustration only and notintended to limit the scope of the invention in any way.

Formulation Example

Hard gelatin capsules were prepared using the following ingredients:

    ______________________________________                                                              Quantity                                                                      (mg/capsule)                                            ______________________________________                                        Active ingredient (Citrus peel extract)                                                                20                                                   Starch, dried           160                                                   Magnesium stearate       20                                                   Total                   200 mg                                                ______________________________________                                    

While the invention has been described with respect to the abovespecific embodiments, it should be recognized that various modificationsand changes may be made to the invention by those skilled in the artwhich also fall within the scope of the invention as defined by theappended claims.

What is claimed is:
 1. A method for inhibiting the activity of3-hydroxy-3-methylglutaryl CoA(HMG-CoA) reductase in a mammal whichcomprises administering an effective amount of citrus peel extractthereto.
 2. The method of claim 1, wherein the mammal is human.
 3. Themethod of claim 1, wherein the citrus is tangerines, oranges, lemons,grapefruits or Poncirus trifoliata.
 4. The method of claim 2, whereinthe effective amount of citrus peel extract ranges from 10 to 500 mg/kgbody weight/day.
 5. The method of claim 1, wherein said citrus peelextract is administered in the form of a pharmaceutical composition. 6.The method of claim 1, wherein said citrus peel extract is administeredin the form of an additive or a dietary supplement in food or beverage.